Retromer deficiency in Tauopathy models enhances the truncation and toxicity of Tau

Alteration of the levels, localization or post-translational processing of the microtubule associated protein Tau is associated with many neurodegenerative disorders. Here we develop adult-onset models for human Tau (hTau) toxicity in Drosophila that enable age-dependent quantitative measurement of central nervous system synapse loss and axonal degeneration, in addition to effects upon lifespan, to facilitate evaluation of factors that may contribute to Tau-dependent neurodegeneration. Using these models, we interrogate the interaction of hTau with the retromer complex, an evolutionarily conserved cargo-sorting protein assembly, whose reduced activity has been associated with both Parkinson’s and late onset Alzheimer’s disease. We reveal that reduction of retromer activity induces a potent enhancement of hTau toxicity upon synapse loss, axon retraction and lifespan through a specific increase in the production of a C-terminal truncated isoform of hTau. Our data establish a molecular and subcellular mechanism necessary and sufficient for the depletion of retromer activity to exacerbate Tau-dependent neurodegeneration.

Supplementary Methods 1

Eye phenotypes and scoring
All flies were reared at 25°C and eyes analysed within 2 days after eclosion. GMR-Gal4 was used for eye expression of transgenes. Adult eyes were imaged and recorded using a Leica MZ 16 1FA stereo microscope and scored blind to genotype, as described previously 8 with modifications to the scoring criteria as follows: (0) wild-type, (1) mild roughness and distinguishable malformed and fused ommatidia with size reduced to ≥ 60% of that of wild-type with occasional black lesions, (2) more pronounced reduction of size (≥ 50% of wild-type), more frequent black lesions and obvious ommatidia malformation and fusion, (3) size reduced to ≥ 35% of wild-type, have frequent black lesions and difficult to distinguish fused ommatidia boundries, (4) narrow 'bar-shaped' eyes (≥ 25% of wild-type size), severe ommatidia fusion with most boundaries undistinguishable and frequent large black lesions, (5) eye size severely reduced (≥ 15-20% of wild-type), no observable ommatidia boundaries, consistent occurrence of large black lesions, (6) very small and severely malformed eyes (≥ 10% wild-type size), complete disruption of organization with no distinguishable boundaries, often consisting of single black lesion. A minimum of 20 individual animals were scored per group.
All scoring data are provided in Table S2.

Scanning Electron Microscopy
Animals were anesthetized with CO2 and fixed with 2.5% glutaraldehyde for two hours at room temperature. Preparations were then washed three times with 0.1M cacodylate buffer for two minutes each and postfixed with 1% osmium tetroxide for thirty minutes. The samples were washed twice with H2O for three minutes each, and dehydrated through an ethanol series (1x30%, 1x50%,1x 70%,1x 90%, 1x95% and 2x100%). The preparations were then dried at critical point and sputtercoated with 10 nm gold-palladium and imaged with a Zeiss Merlin Scanning Electron Microscope. Results were similar to 6x-mCherry soma count.

Cell loss assays
Adult and larval brain immunostaining Adult brain dissections were performed according to Williamson et al. 20 In brief, adult brains were dissected in PBS solution and were then transferred to 4% formaldehyde in PBS and fixed overnight at 4°C. The next day, the brains were permeabilized in 0.3% PBST [PBS + Triton] and were incubated with primary antibodies for 3 days and were then incubated probed with secondary antibodies overnight. Labelled brains were mounted in ProLong Gold (Thermo Fisher). Quantification of DC neuron medulla axon coverage was performed using the threshold tool in ImageJ 21 . Briefly, medulla areas were cropped from Z projections of brain images. Pixel intensity was then set such that axons were above threshold. The percent medulla area occupied by axons was then measured.

Supplementary Methods 3
Data are provided in Table S2. Larval brain dissections were performed essentially as described elsewhere 22 with the following modifications: larvae were dissected in cold PBS, fillets were prepared without removing the brain and fixed in Bouin's solution for 5 minutes. After staining with primary and secondary antibodies, the brains were dissociated from the carcass and mounted in ProLong Gold (Thermo Fisher). For staining of truncated hTau (hTau-421D), we empirically found 0.1% PBST for incubation and washes avoided disruption of intracellular organelles including endosomes. The